Plastid marker-gene excision by transiently expressed CRE recombinase
Article first published online: 10 JAN 2006
The Plant Journal
Volume 45, Issue 3, pages 447–456, February 2006
How to Cite
Lutz, K. A., Bosacchi, M. H. and Maliga, P. (2006), Plastid marker-gene excision by transiently expressed CRE recombinase. The Plant Journal, 45: 447–456. doi: 10.1111/j.1365-313X.2005.02608.x
- Issue published online: 10 JAN 2006
- Article first published online: 10 JAN 2006
- Received 16 June 2005; revised 13 September 2005; accepted 3 October 2005.
- plastid transformation;
- marker-gene excision;
- CRE-loxP site-specific recombination;
- transient gene expression
We report plastid marker-gene excision with a transiently expressed CRE, site-specific recombinase. This is a novel protocol that enables rapid removal of marker genes from the approximately 10 000 plastid genome copies without transformation of the plant nucleus. Plastid marker excision was tested in tobacco plants transformed with a prototype polycistronic plastid vector, pPRV110L, designed to express multiple genes organized in an operon. The pMHB10 and pMHB11 constructs described here are dicistronic and encode genes for herbicide (bar) and spectinomycin (aadA) resistance. In vector pMHB11, expression of herbicide resistance is dependent on conversion of an ACG codon to an AUG translation initiation codon by mRNA editing, a safety feature that prevents translation of the mRNA in prokaryotes and in the plant nucleus. In the vectors, the marker gene (aadA) is flanked by 34-bp loxP sites for excision by CRE. Marker excision by a transiently expressed CRE involves introduction of CRE in transplastomic leaves by agro-infiltration, followed by plant regeneration. In tobacco transformed with vectors pMHB10 and pMHB11, Southern analysis and PCR identified approximately 10% of the regenerated plants as marker-free.